Abstract The proposed studies will advance the understanding of enteric disease caused by food-borne, Shiga-toxin- producing enterohemorrhagic E. coli (EHEC), whichis the major cause of life-threatening hemorrhagic colitis and hemolytic uremic syndrome in the US. While Shiga toxin 2a is the main virulence factor for intestinal and extra-intestinal disease manifestations, clinical data strongly suggest that Stx-toxemia occurs early in illness and is short-lived. Thus, our studies will focus on early EHEC-host interactions, particularly the complex interaction between EHEC and the human colonocyte, goblet cells and immune cells, including macrophages and neutrophils. Our exciting preliminary data are gained using a novel model of EHEC infection, termed human colonoid monolayers (HCM), which are primary colonic epithelial cultures derived from adult stem cells isolated from colonic biopsies of healthy donors. Upon differentiation, HCM are composed of all major types of epithelial cells, including colonocyte, goblet and enteroendocrine cells. They develop mature microvilli and produce a thick layer of mucus similar to that which is normally present in the human colon. Using these HCM we are demonstrating here that EHEC infection results in the destruction of the mucus layer allowing EHEC to gain access to the colonocyte surface. The serine protease EspP plays a role in EHEC colonization and also induces the ion secretion, indicating its role in EHEC-induced watery diarrhea. These data suggest that human colonoids recapitulate EHEC infection and provide unique insights into pathogenesis that differ from other studies performed in human colon cancer cell lines, allowing improved appreciation of the roles of virulence factors and assessment of novel therapeutic targets. Relevant to this pathogen, we will test the hypothesis that HCM, which uniquely represent ex vivo human colonic epithelium, recapitulate human EHEC infection and provide unique insights in pathogenesis. To test this hypothesis and enhance our understanding of EHEC intestinal pathogenesis, we propose the following Aims: 1. Determine the role of serine protease EspP in EHEC colonization of human colonic epithelium and virulence development. 2. Determine the molecular mechanisms of EHEC induced watery diarrhea. 3. Determine the role of macrophages and neutrophils in EHEC-induced immune response. The results gained from the proposed experiments will further elucidate the molecular mechanisms for EHEC interactions with human colonic epithelium, test the role of EspP serine protease in EHEC colonization and development of watery diarrhea as well as establish the role of neutrophil and macrophages in EHEC clearance.